Phage display has been known and widely applied in the biological sciences and biotechnology (see U.S. Pat. Nos. 5,223,409; 5,403,484; 5,457,1698; 5,766,905; and the references cited therein). The methodology utilizes fusions of nucleic acid sequences encoding foreign polypeptides of interest to sequences encoding phage coat proteins to display the foreign polypeptides on the surface of bacteriophage particles. Applications of the technology include the use of affinity interactions to select particular clones from a library of polypeptides, the members of which are displayed on the surfaces of individual phage particles. Display of the polypeptides is due to expression of sequences encoding them from phage vectors into which the sequences have been inserted. Thus a library of polypeptide encoding sequences are transferred to individual display phage vectors to form a phage library that can be used to screen for polypeptides of interest.
Phage display has been used in a variety of ways and has also been modified to facilitate the isolation of the displayed polypeptide. Ward et al. (J. Imm. Meth. 189(1):73-82, 1996) describe the introduction of sequence encoding an enzymatic cleavage site between sequences encoding a human IgG1 polypeptide and a truncated M13 phage gene III. After expression on a phage surface, the polypeptide was separable from the phage by enzymatic cleavage.
Phage display based upon filamentous bacteriophage fd has also been modified to utilize sequences encoding a heterologous polypeptide and a sequence encoding a phage protein such that expression of the polypeptide may be in a soluble form or as a fusion with the phage coat protein depending upon the cell line used (see Hoogenboom et al., Nucl. Acids Res. 19(15):4133-7, 1991, and Lucic et al., J. Biotech. 61:95-108, 1998). Similarly modified sequences have been used in bacteriophage λ based display systems to conditionally express heterologous polypeptides on bacteriophage λ heads (see Mikawa et al., J. Mol. Biol. 262:21-30, 1996).
A constraint associated with phage display, however, is where expression of a heterologous polypeptide affects the viability of the host cell used to propagate the phage library or used to produce phage for display. One approach to address this constraint has been by the use of a tightly regulated promoter to control the expression of fusions of a heterologous polypeptide and a phage coat protein, and thus control display of proteins on phage (see Huang et al. Gene, 251:187-197, 2000). This approach does not fully address a second difficulty, however, where the presence a heterologous polypeptide as a fusion with a phage coat protein results in interference with the phage life cycle. A possible approach to address both interference with phage life cycle and negative effects on host cell viability is to use modified regulators of transcription and/or translation that decrease the level of expression of the heterologous polypeptide.
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